Checklist and phenetics studies of nymphs of two species of triatomines: Triatoma lenti Sherlock & Serafim, 1967 and Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira, Barata, 2002 (Hemiptera: Reduviidae: Triatominae)

Abstract INTRODUCTION: Triatoma lenti and Triatoma sherlocki are endemic species of the State of Bahia, located in northeastern Brazil, where they have records of domiciliation in the human environment. In view of the epidemiological aspect and taxonomic importance of these species for the systematics of the Triatoma genus, a study was carried out with nymphs of all five instars. METHODS An extensive review of studies on nymphs from the subfamily Triatominae is presented. Morphology was studied using a scanning electron microscope and morphometric analyses. RESULTS: The morphological study allowed us to characterize and discriminate species by means of scanning electron microscope of the last abdominal segment. In addition, the results show morphometric variability, with the total size of the head that best discriminates the species. CONCLUSIONS: Studies on nymphs are fundamental to the ecosystem; however, the literature on the immature forms of certain groups is scarce, difficult to use, or nonexistent. Therefore, this study includes morphological and morphometric data of the nymphal instars of T. lenti and T. sherlocki, corroborating the specific taxonomy of these species.


INTRODUCTION
In the Americas, various species of triatomines are vectors of Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae), the etiological agent of Chagas disease 1 . The insects of the subfamily Triatominae (Jeannel, 1919) are hematophagous and feed primarily on vertebrate blood 2 . Even though all species of triatomines are hematophagous, species that colonize residential places or are peridomicile have increased chances of transmitting T. cruzi to humans 3 . After the successful Triatoma infestans (Klug, 1834) control program conducted by the Brazilian National Health Foundation, other triatomines previously considered predominately sylvatic have emerged as potential vectors in several areas of Brazil 4,5 .
Morphology and morphometry are tools that contribute to the knowledge of triatomines and generate useful information to establish more effective strategies for vector control 17 . In Triatominae, biometric studies are used to characterize new species, detect populations, and define structures 18 . For example, geometric morphometry allows the collection of information about the shape and size of organisms, which helps in systematic and phylogenetic studies 10,19,20 .
Several authors have used morphology and morphometry to characterize the species and correlate the known characteristics of the character, isoenzymatic and ecological, and contributed to both systematic analyses. Studies on immature instars of T. lenti and T. sherlock are scarce; therefore, we evaluated and characterized those species that make up the T. brasiliensis subcomplex, a relevant group for the ecoepidemiology of Chagas disease in the northeastern region of Brazil [21][22][23][24][25] , by gathering all information from the literature on the study of immature forms in Triatominae.

Insects
We used specimens from a T. lenti (Figure 1) colony collected on April 9, 2008, which were found in the county of Macaúbas

Morphological analyses
The fifth instar nymphs from T. sherlocki and T. lenti ( Figure  1) were cleaned using an ultrasound device. Next, the structures were dehydrated in alcohol, dried in an incubator at 45ºC for 20 min, and fixed in small aluminum cylinders with transparent glass. Sputtering metallization was then performed on the samples for 2 min at 10 mA in an Edwards sputter coater. After metallization, the samples were studied and photographed using a Topcon SM-300 scanning electron microscope (SEM), according to Rosa et al. 26 . The images obtained were processed (background, contrast, brightness) using the GNU Image Manipulation Program v2.0.2 (GIMP) software free and open-source image editor, and the structures were described and compared.

Morphometric analyses
For the T. lenti and T. sherlocki measurements, 15 nymphs specimens in the first, second, third, fourth, and fifth instars were fixed on glass slides using a double-sided tape. Measurements were also taken to determine the thorax, abdomen, and head length, as well as interocular, ante-ocular, and postocular distance, eye diameter, and the three proboscis segments. These distances were defined by Dujardin et al. 27 . The measurements were taken using a Leica MZ APO stereomicroscope and analyzed using the Motic Advanced 3.2 image analysis software. Descriptive statistics analyses and Welch's t-test were performed using GraphPad Prism v.5.03.

Principal component analysis
To visualize the general patterns of morphological variation in the multidimensional data obtained with the principal component analysis (PCA) of the references, a factorial map was generated using Past 3.2 28 .

RESULTS
Through an extensive literature survey on immature forms of triatomines, we recovered 115 studies that explored the morphological aspects of nymphs; therefore, we updated the list presented by Galvão 2014 6 ( Table 1).

Morphological characteristics
The morphological characteristics of the two species are presented in Figure 2. According to the genital morphology of fifth instar nymphs, the ninth ventral abdominal segment is wider in T. lenti than in T. sherlocki, as well as the presence of a hole in the posterior portion of this segment in T. lenti and its absence in T. shelocki. The ninth ventral abdominal segment of fifth instar nymphs shows parallel grooves in the posterior region, which are most evident in T. lenti, while their presence in T. sherlocki is poorly visible. The eighth segment was trapezoidal in T. sherlocki and oval in T. lenti. The laterals were irregular at the apex. It was found that the ninth segment had few sensilla, as well as segments

Triatoma infestans
Sexual distinction between 5 th instar nymphs by SEM Morphology of 5 th instar nymphs by SEM Abdominal structures of 5 th stage nymphs Description of nymphs of 1 st , 2 nd , 3 rd , 4 th , and 5 th instars (MO) Rosa et al. 26 Rosa et al. 42 Rosa et al. 43 Galíndez-Girón et al. 35

Triatoma jurbergi
Description of nymphs of 1 st , 2 nd , 3 rd , 4 th , and 5 th instars (MO, MEV) Comparative study of stridulatorium sulcus, bucculla and rostrum Jurberg et al. 66 Silva et al. 67 Triatoma klugi Description of nymphs (MO) and visualization of structures using SEM. Comparative study of the stridulatorium sulcus, buccula and rostrum of nymphs

Triatoma matogrossensis
Abdominal structures of 5 th instar nymphs Sexual distinction between 5 th instar nymphs Morphology of 5 th instar nymphs by SEM Rosa et al. 26 Rosa et al. 42 Rosa et al. 43 Triatoma melanocephala Morphometric characterization of the nymphal instars Oliveira et al. 68 Jurberg

Triatoma rubrovaria
Morphology of the head of 1 st and 5 th instar nymphs Antenna morphometry Morphological and key (MO and SEM) Rosa et al. 56 Rosa et al. 57 Galíndez-Girón et al. 35

Triatoma tibiamaculata
Abdominal structures of 5-instar nymphs Rosa & Barata,44 Triatoma vandae Description of nymphs (MO) and visualization of structures using SEM. Comparative study of the stridulatorium sulcus, buccula and rostrum of nymphs Silva et al. 72 Silva et al. 67

Triatoma vitticeps
Antenna morphometry and morphology Morphology compared to other Reduviidae Rosa et al. 45 Weirauch 73

Triatoma williami
Description of nymphs (MO) and visualization of structures using SEM.

Morphometric characteristics
Morphometric characteristics of the two species are presented in Table 2. The averages of the total length of heads in T. sherlocki and T. lenti were 1. 34  The averages of the total length of thoraxes in T. sherlocki and T. lenti were 0.98 and 0.99 mm, 1.43 and 1.43 mm, 2.08 and 2.02 mm, 2.93 and 3.26 mm, and 4.68 and 5.29 mm in the first, second, third, fourth, and fifth instar, respectively.
The averages of the total length of abdomens in T. sherlocki and T. lenti were 1.75 and 1.57 mm, 3.17 and 3.04 mm, 5.24 and 5.60 mm, 6.68 and 6.63 mm, and 11.29 and 11.14 mm in the first, second, third, fourth, and fifth instar, respectively. The mean lengths of the abdomen were larger than those of the head, which were larger than those of the thorax in the first stage nymphs in both T. lenti and T. sherlocki. As in the first stage nymphs, the average abdominal length in second instar nymphs was longer than those of the head and the thorax nymphs for both species.
In T. lenti and T. sherlocki, the highest measurement observed was the total length of the abdomen that was longer than the head as well as the thorax. The average lengths of the abdomen were higher than those of the head as well as those of the thorax for T. lenti and T. sherlocki. Abdomen and eye diameter measurements showed no significant difference between T. lenti and T. sherlocki (Table 2. p<0.001). Analyzing the fifth instar nymphs of the two species, we found that the abdomen was the largest segment, and unlike the other nymphal instars, the thorax was larger than the head in the fifth stage nymphs of both species.
The PO, IE, and AO followed an ascending order: first instar > second instar > third instar > fourth instar > fifth instar, for both species (Table 2). Among these parameters, the largest length was 8/12 the distance before the eye, and the smallest was the diameter of the eyes and the distance between both T. lenti and T. sherlocki.
The first, second, and third proboscis segment lengths were in the following order: first segment > third segment > second ( Table 2). In fifth stage nymphs, the second and third segments showed significant differences in their length for both species. In both cases, it was observed that the second segment was larger than the third and this was larger than the first for all nymphal instars (Table 2). After measuring and performing statistical analysis on the three segments of the proboscis in first stage nymphs, it was observed that only the second segment showed a significant difference, while the first and third segments did not show significant differences between T. lenti and T. sherlocki (Table 3).
Comparisons between proboscis segments and head and abdomen lengths of the two species are presented in Table 3. In the second instar nymphs, the three segments of the proboscis revealed measurements that showed significant differences, according to the statistical analysis, for the two species. In the third and fourth instar nymphs, measurements of the second and third proboscis segments showed a significant difference between T. lenti and T. sherlocki.
In the first instar nymphs, statistical analyses revealed significant differences in total head and abdominal length measurements. Thorax measurements were not different between the two species. Regarding the measurements of head parameters of the first stage nymphs, the distance between the anterior, postocular, interocular, and eye diameters were significantly different when comparing T. lenti and T. sherlocki. Statistical analysis showed significant differences only for interocular distance and eye diameter in second instar nymphs.
Measurements of the thorax, abdomen, ante-ocular distance, postocular distance, and total head length revealed no significant differences between the two species (Table 3). For the third instar nymphs, all measurements except for the eye diameter and first proboscis segment measurements, showed significant differences between both species, ( Table 3). For the fourth instar nymphs, all parameters showed statistically significant differences, except for the eye diameter and first segment of the proboscis, as was also observed for the third instar nymphs ( Table 3). The measurements of total head length, ante-ocular distance, postocular distance, and interocular and thorax distance of fifth instar nymphs were significantly different between the two species.

Principal component analysis
The main components (PCA1 and PCA2) are presented through biplot graphics showing the morphometric variability between T. lenti and T. sherlocki. The total size of the head was responsible for greater discrimination between the studied specimens. Alternatively, PC1 and PC2 were 99.569% and 0.431% for the first stage nymphs (Supp. Figure 1), 99.966% and 0.034% for the second stage (Supp. Figure 2), 99.937% and 0.062% for the third stage (Supp. Figure 3), 99.791% and 0.208% for the fourth (Supp. Figure 4), and 99.84 and 0.15% for the fifth (Supp. Figure 5).

DISCUSSION
Studies on immature forms of triatomines are relevant to taxonomy and provide important information for the understanding of several biological aspects of these vectors. In this study, a list of works with immature forms were presented and a morphological characterization of five nymphal instars of T. lenti and T. sherlocki, species that are closely related phylogenetically 10,74 , were described.

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Triatoma lenti and T. sherlocki have reproductive compatibility with other members of the species T. brasiliensis subcomplex 5 , which are frequently found in dwellings and infected with T. cruzi; therefore, they are potential vectors of Chagas disease 3,75 . Costa et al. 76 conducted a comparative morphological analysis of the external genital structures and eggs of T. brasiliensis to differentiate chromatic forms. Gonçalves et al. 77 used classic and geometric morphometry as a tool to distinguish T. jatai from other species. Mendonça et al. 14 , used morphological, morphometric, molecular, and cytogenetic approaches as well as experimental crosses to revalidate the specific status of T. bahiensis and differentiate it from T. lenti. Combining morphometric and molecular approaches has provided important clues about the T. brasiliensis complex, which includes the species and subspecies T. lenti, T. petrocchiae, T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis, T. sherlocki, T. melanica, and T. bahiensis 10 .
In the present study, using SEM images, morphological differences were observed in the ninth ventral abdominal segment of female and male nymphs of the fifth instar. Comparing the morphology of the ninth ventral abdominal segment of male and female nymphs in the fifth instar of the species T. melanocephala Neiva & Pinto, 1923, T. brasiliensis, T. infestans, T. matogrossensis Leite and Barbosa, 1953, T. tibiamaculata (Pinto, 1926), T. lenti, and T. sherlocki, it can be seen that these seven species differ by this character 26,42,78 . This indicates that the shape and size of the ninth abdominal segment in fifth instar nymphs may be taxonomically valid.
The measurements of the head, thorax, and abdomen served to better characterize and distinguish T. lenti and T. sherlocki across their evolutionary instars, as well as in the comparative analysis of nymphal instars of other species of the Triatoma genus. The combination of morphometric and morphological approaches provides important clues about the delimitation of the complex 26,76,79 . Oliveira et al. 10 morphometrically analyzed the species of the T. brasiliensis complex and showed that the variations in the shape of the head were statistically significant. The wings showed sexual dimorphism in shape, while the heads were not dimorphic as expected.
In this study, as in all other nymphal instars, we found that the largest measurement among the head measurements was the anocular distance and the smallest was the postocular distance, In the morphometry, all parameters in the first instar, except the average eye diameter and the first and third proboscis segments, were significantly different between T. lenti and T. sherlocki. Measurements of interocular distance, eye diameter, and the three segments of the proboscis revealed significant differences between the second instar nymphs of T. lenti and T. sherlocki. The third and fourth instar nymphs showed significant differences in the measurements of the abdomen, head, thorax, ante-ocular, interocular, postocular, and second and third proboscis segments. In the fifth instar, measurements of thorax length, head length, ante-ocular, interocular, postocular, and second and third proboscis segments showed significant differences in taxonomic differentiation between T. lenti and T. sherlocki. In all nymphal instars, the total length measurement ratio were in the following order: abdomen > head > thorax. In Triatoma melanocephala Neiva & Pinto, 1923, the nymphal instars presented the following length pattern: in the first instar, thorax > abdomen > head; in the second instar, abdomen > head > thorax; and in the third, fourth, and fifth instars, abdomen > thorax > head 81 .
In all nymphal instars of T. lenti and T. sherlocki, it was observed that the second segment of the proboscis was larger than the third which was larger than the first segment. In T. melanocephala nymphs, it was found that specimens in the first three nymphs presented the same length order (2 > 3 > 1), while those in the fourth and fifth instars, along with the adults, possessed mouthpart segments of the same order (2 > 1 > 3) 78 . The main components (PCA1 and PCA2) illustrated the differences between the studied parameters and showed that the total size of the head is or that it discriminates against T. lenti and T. sherlocki.
Studies on nymphs are crucial for the systematic development of certain groups. However, the literature on immature forms of certain groups is scarce, difficult to use, or nonexistent 79 . Epidemiological studies and control measures require precise taxonomic determination of T. brasiliensis subcomplex 80,86,89 . Therefore, this study provides morphological and morphometric data on the nymphal instars of T. lenti and T. sherlocki, corroborating the specific taxonomy of these species.

ACKNOWLEDGMENTS
The authors are thanks to Capes (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Thanks to the São Paulo State University (Unesp), Faculty of Pharmaceutical Sciences. The authors also thank the researchers and students of the Parasitology Laboratory at the same institution. We thank the members of the Institute of Biology at the State University of Campinas (Unicamp).

AUTHORS' CONTRIBUTION
JAR: Conception, design study and data acquisition. VFP: Writing, Formal analysis and investigation, review, and editing. TB: Writing, Methodology, review, editing, Formal analysis, and investigation. JO: Writing, data acquisition, review, and editing. VJM: Review, data acquisition and editing. LAR: Supervision, Conception, and design study.